Penicillium verruculosum WA 30 a new source of cellulase

Summary Of fungi 110 strains were screened for extracellular cellulase production in shake flask experiments. Twelve strains produced the enzyme in significant quantity. Since the enzyme activity was assayed by different methods (liberation of reducing sugar from cotton, filter paper, carboxymethylcellulose and cellobiose), the estimation of the productivity of the strains differed according to the substrate used. The best cotton degrading activity per fermentation volume as well as per mg of secreted soluble protein was achieved by Penicillium verruculosum WA 30, a wild-type strain, for which the cellulase productivity has not yet been published. The cotton degrading (so-called C₁) activity was successfully enhanced nearly threefold in medium experiments. Analyses of saccharification digests showed that glucose was the predominant product, with negligible amounts of cellobiose. The pH and temperature optima for WA 30 cellulase complex were pH 4.2 and 60°C.     

匍枝根霉纤维二糖合成酶胞内糖基供体的初探及结构功能研究

纤维二糖可有效诱导丝状真菌产纤维素酶,前期研究表明匍枝根霉Rhizopus stolonifer TP-02具有纤维二糖合成酶(CBS),可以尿苷二磷酸葡萄糖(UDPG)为糖基供体合成纤维二糖,从而开启纤维素酶的自诱导合成途径。为研究R. stolonifer中纤维二糖的胞内合成途径,通过重叠PCR在GDP-葡糖焦磷酸化酶基因ggp中引入硫胺吡啶抗性基因ptrA,分别转化原菌TP-02和△ugp突变株,构建△ggp和△ugp/△ggp突变株。利用液质联用(LC-MS)检测突变株的胞内糖组分,发现ggp的缺失对胞内纤维二糖合成的影响较弱,但同时缺失ugp则将直接导致二糖合成受阻。RT-qPCR结果显示△ggp突变株中纤维素酶基因转录水平较原株TP-02下调20%左右,而△ugp/△ggp突变株中被测基因的转录水平则出现了高达80%左右的下调。同时对突变株纤维素酶表达水平进行研究,发现△ugp/△ggp突变株中几乎检测不到纤维素酶活力。结果显示,UDPG为R. stolonifer胞内合成纤维二糖的主要糖基供体,而GDPG可能是UDPG的替代物,在UDPG不足时维持胞内二糖合成。此外,利用生物信息学方法对CBS结构功能深入分析,经丙氨酸扫描确定其合成纤维二糖的关键作用残基为Asp210和Asp300,为后续进一步研究及理性改造提供方向和理论依据。     

Isolation and partial properties of cellulose-decomposing strain of Cytophaga sp. LX-7 from soil

A new facultatively anaerobic, Gram-negative bacterium, Cytophaga sp. LX-7, degrading crystalline cellulose completely, was isolated from soil by dilution plating on cellodextrin agarose plates. This strain could excrete extracellularly all three types of cellulase and cellulosic substrates were the strongest inducer of endocellulase with CMC-liquefying activity production. No reducing sugar was found in cultures of cellulose during incubation. An enzyme which degrades crystalline cellulose was detected in cultures of cellulose by measuring the formation of soluble carbohydrate but was not detected by determining the reducing sugar released. This strain also synthesized cell-bound cellobiose oxidizing enzyme which was previously noted only in fungi. Both cellulose and soluble sugars could promote the production of cellobiose oxidizing enzyme.     

A Comparison between the Cellulolytic Activity of White and Brown Rot Fungi. I. The Activity on Insoluble Cellulose

About 70 strains of white and brown rot fungi were cultivated on media, containing filter paper cellulose as the main carbon source. The cellulolytic activity of the culture filtrates was measured after different periods of growth by means of the turbidimetric method. The results obtained indicate a difference between the two types of wood decay fungi as to the capacity of attacking the cellulose used in the medium and in the cellulase test. No significant C₁activity was found in any of the brown rot cultures whereas all white rot fungi tested exerted a measurable activity on the test substrate. The effect of various carbohydrates and some proteins as inducers of cellulase activity was studied. Especially cellobiose and lactose were active on white rot fungi in this respect, particularly in the presence of yeast extract. Also some brown rot fungi exerted C₁-activity after incubation on glucose or cellobiose.     

Yersinia kristensenii: A new species of enterobacteriaceae composed of sucrose-negative strains (formerly called atypicalYersinia enterocolitica orYersinia enterocolitica-Like)

Induction of cellulase was observed inFusarium sp. with reduction in lag period by lactose-pregrown cells as compared with glucose-pregrown cells. Insoluble cellulose (Sigmacell) induced maximum cellulase production in the induction medium. Supplementation of the culture growing on cellulose by cellobiose or glucose resulted in increased cellular growth and decreased cellulase production. Stepfeeding of cellobiose to the culture growting on carboxymethyl cellulose resulted in decreased cellulase production. Significant cellulase activity was detected in the culture filtrate of cells growing on Sigmacell supplemented with glucose, only when the glucose disappeared from the medium. This suggests that cellulase production may in part be regulated by catabolite repression.     

The cellobiose-oxidizing enzymes CBQ and CbO as related to lignin and cellulose degradation — a review

Abstract: In this review properties of cellobiose:quinone oxidoreductase (CBQ) and cellobiose oxidase (CbO) are presented and their possible involvement in lignin and cellulose degradation is discussed. Although these enzymes are produced by many different fungi, their importance for wood-degrading fungi is the topic here. CBQ is a FAD enzyme, while CbO also contains a heine group of the cytochrome b type. Protease activity is reported to convert CbO to CBQ. During oxidation of cellobiose (emanating from cellulose) to cellobiono-l,5-lactone, both enzymes reduce quinones produced by laccase and peroxidase during lignin degradation to the corresponding phenols. Many phenoxy and cation radicals are also reduced. Quinone reduction is more rapid than oxygen reduction, although oxygen is slowly reduced to superoxide and/or hydrogen peroxide. Thus, a more appropriate name for CbO is cellobiose dehydrogenase. CbO also reduces Fe(III) and together with hydrogen peroxide produced by the enzyme Fenton's reagent may be formed, resulting in hydroxyl radical production. This radical can degrade both lignin and cellulose, possibly indicating that cellobiose oxidase has a central role in degradation of wood by wood-degrading fungi.     

β-Glucanase expression by Ruminococcus flavefaciens FD-1

Abstract Ruminococcus flavefaciens has been hypothesized to produce cellulase constitutively. We have studied the effect of carbon source, either cellobiose or cellulose, on the production of cellulase in batch cultures of R. flavefaciens FD-1. Total CMCase and ¹⁴C-cellulase activity was approximately 2-fold higher in cellobiose grown cells than in cellulose grown cells, whereas p-nitrophenyl-β- d -cellobiosidase (PNPCase) activity was not affected by culture conditions. The addition of cellulose to cells growing on cellobiose did not alter the amount or rate of PNPCase and ¹⁴C-cellulase production. Northern blot analysis of mRNAs produced by R. flavefaciens FD-1 grown using either cellobiose or cellulose as the substrate indicated that two of the four β-glucanase genes cloned from R. flavefaciens FD-1 were only expressed in cells grown with cellulose as the substrate. Although the adherence of cells and cellulase enzyme to native cellulose can complicate interpretations of these data, the results indicate that cellulase synthesis by R. flavefaciens is differentially regulated by carbon source.     

Enhanced production of cellulase usingAcidothermus cellulyticus in fed-batch culture

Summary Fed-batch fermentations of Acidothermus cellulolyticus utilizing mixtures of cellulose and sugars were investigated for potential improvements in cellulase enzyme production. In these fermentations, we combined cellulose from several sources with various simple sugars at selected concentrations. The best source of cellulose for cellulase production was found to be ball-milled Solka Floc at 15 g/l. Fed-batch fermentations with cellobiose and Solka Floc increased cell mass only slightly, but succeeded in significantly enhancing cellulase synthesis compared to batch conditions. Maximum cellulase activities obtained from fermentations initiated with 2.5 g cellobiose/l and 15 g Solka Floc/l were 0.187 units (U)/ml, achieved by continuous feeding to maintain <0.1 g cellobiose/l, and 0.215 U/ml using the same initial medium when 2.5 g cellobiose/l was step-fed after the sugar was nearly consumed. In batch, dual-substrate systems consisting of simple sugars with Solka Floc, substrate inhibition was evident in terms of specific growth rates, specific productivity values, and maximum enzyme yields. Limiting concentrations of glucose or sucrose at 5 g/l, and cellobiose at 2.5 g/l, in the presence of Solka Floc, yielded cellulase activities of 0.134, 0.159, and 0.164 U/ml, respectively. Offprint requests to: M. E. Himmel     

Evidence for Transceptor Function of Cellodextrin Transporters in Neurospora crassa

Neurospora crassa colonizes burnt grasslands and metabolizes both cellulose and hemicellulose from plant cell walls. When switched from a favored carbon source to cellulose, N. crassa dramatically up-regulates expression and secretion of genes encoding lignocellulolytic enzymes. However, the means by which N. crassa and other filamentous fungi sense the presence of cellulose in the environment remains unclear. Previously, we have shown that a N. crassa mutant carrying deletions of three β-glucosidase enzymes (Δ3βG) lacks β-glucosidase activity, but efficiently induces cellulase gene expression and cellulolytic activity in the presence of cellobiose as the sole carbon source. These observations indicate that cellobiose, or a modified version of cellobiose, functions as an inducer of lignocellulolytic gene expression and activity in N. crassa. Here, we show that in N. crassa, two cellodextrin transporters, CDT-1 and CDT-2, contribute to cellulose sensing. A N. crassa mutant carrying deletions for both transporters is unable to induce cellulase gene expression in response to crystalline cellulose. Furthermore, a mutant lacking genes encoding both the β-glucosidase enzymes and cellodextrin transporters (Δ3βGΔ2T) does not induce cellulase gene expression in response to cellobiose. Point mutations that severely reduce cellobiose transport by either CDT-1 or CDT-2 when expressed individually do not greatly impact cellobiose induction of cellulase gene expression. These data suggest that the N. crassa cellodextrin transporters act as “transceptors” with dual functions - cellodextrin transport and receptor signaling that results in downstream activation of cellulolytic gene expression. Similar mechanisms of transceptor activity likely occur in related ascomycetes used for industrial cellulase production.     

On the mode of action of fungal cellulases

Summary The mode of action of the cellulolytic enzymes of two strong cellulose decomposing fungi, Penicillium oxalicum Curie et Thom and Helminthosporium cyclops Drechsler, was studied. The culture filtrates and enzyme preparations obtained from them showed high cellulase activity and very weak cellobiase activity. The cellulolytic system of both experimental organisms seems to be multicomponent. The cellulase component showed its activity mainly extracellulary and the cellobiase component, mainly intracellulary. It seems, therefore, that during growth of both fungi on a cellulose medium, the extracellular cellulase acts hydrolytically on the cellulose substrate forming cellobiose which is further acted upon by intracellular cellobiase to form glucose. Paper chromatographic assay of the products of the enzymatic reaction sub-stantiated this conclusion.     

Production of cellobiose by enzymatic hydrolysis: removal of beta-glucosidase from cellulase by affinity precipitation using chitosan

Removal of beta-glucosidase (BG) from cellulase is essential to the enzymatic production of cellobiose from cellulose because of the high reactivity of BG with cellobiose to form glucose. Chitosan is a reversibly soluble-insoluble polymer depending on pH, and it has an affinity with the other components, endo-beta-1,4-glucanase and cellobiohydrolase, or cellulase. The affinity precipitation technique using chitosan is an effective way to fractionate cellulase for the above purpose. Hydrolysis experiments of cellulose with the residual fractionated enzyme gave higher cellobiose contents in the soluble sugar products. (c) 1993 John Wiley & Sons, Inc.     

Effects of cellobiose and glucose on cellulose hydrolysis by both growing and resting cells of Bacteroides cellulosolvens

The cellulase system of Bacteroides cellulosolvens was subjected to both catabolite repression and feedback inhibition by cellobiose. Cellulose-solubilizing activity was 50% inhibited at a cellobiose concentration of 2.6 g/L and completely inhibited by 12 g/L. Glucose at 12 g/L (the highest concentration tested) had no effect on cellulase activity. Supplementation of B. cellulosolvens cellulase with beta-glucosidase resulted in increased conversion of cellobiose to glucose; however, a constant cellobiose pool size of approximately 7 g/L was maintained.     

Differential Involvement of β-Glucosidases from Hypocrea jecorina in Rapid Induction of Cellulase Genes by Cellulose and Cellobiose

Appropriate perception of cellulose outside the cell by transforming it into an intracellular signal ensures the rapid production of cellulases by cellulolytic Hypocrea jecorina. The major extracellular β-glucosidase BglI (CEL3a) has been shown to contribute to the efficient induction of cellulase genes. Multiple β-glucosidases belonging to glycosyl hydrolase (GH) family 3 and 1, however, exist in H. jecorina. Here we demonstrated that CEL1b, like CEL1a, was an intracellular β-glucosidase displaying in vitro transglycosylation activity. We then found evidence that these two major intracellular β-glucosidases were involved in the rapid induction of cellulase genes by insoluble cellulose. Deletion of cel1a and cel1b significantly compromised the efficient gene expression of the major cellulase gene, cbh1. Simultaneous absence of BglI, CEL1a, and CEL1b caused the induction of the cellulase gene by cellulose to further deteriorate. The induction defect, however, was not observed with cellobiose. The absence of the three β-glucosidases, rather, facilitated the induced synthesis of cellulase on cellobiose. Furthermore, addition of cellobiose restored the productive induction on cellulose in the deletion strains. The results indicate that the three β-glucosidases may not participate in transforming cellobiose beyond hydrolysis to provoke cellulase formation in H. jecorina. They may otherwise contribute to the accumulation of cellobiose from cellulose as inducing signals.     

Cellulase biosynthesis during degradation of cellulose derivatives by Thermomonospora curvata

A variety of commercially used cellulose derivatives were compared with crystalline cellulose as substrates for induction of cellulase biosynthesis in the actinomycete Thermomonospora curvata. Cellulase induction during growth on uncoated cellophane was as rapid as that on crystalline cellulose, but on coated cellophanes, induction was delayed. Susceptibility to enzymatic attack determined the inductive potential of the substrate. Cellulose acetate was a poor substrate because of its extreme recalcitrance to attack. With other cellulose derivatives, soluble sugar accumulation caused a transient repression of cellulase biosynthesis, but the ratio of cellobiose (a cellulase inducer) to glucose (a cellulase repressor) was not a controlling factor. Crystalline cellulose yielded the lowest inducer/repressor sugar ratio (1.1:1 compared to 3.8–4.0:1 for cellulose derivatives), but supported the highest cellulase production. Glucose could not repress cellulase biosynthesis in the presence of cellobiose due to the strong preference for uptake of the disaccharide even by glucose-grown cells.     

Regulation of cellulase synthesis in batch and continuous cultures of Clostridium thermocellum

Regulation of cell-specific cellulase synthesis (expressed in milligrams of cellulase per gram [dry weight] of cells) by Clostridium thermocellum was investigated using an enzyme-linked immunosorbent assay protocol based on antibody raised against a peptide sequence from the scaffoldin protein of the cellulosome (Zhang and Lynd, Anal. Chem. 75:219-227, 2003). The cellulase synthesis in Avicel-grown batch cultures was ninefold greater than that in cellobiose-grown batch cultures. In substrate-limited continuous cultures, however, the cellulase synthesis with Avicel-grown cultures was 1.3- to 2.4-fold greater than that in cellobiose-grown cultures, depending on the dilution rate. The differences between the cellulase yields observed during carbon-limited growth on cellulose and the cellulase yields observed during carbon-limited growth on cellobiose at the same dilution rate suggest that hydrolysis products other than cellobiose affect cellulase synthesis during growth on cellulose and/or that the presence of insoluble cellulose triggers an increase in cellulase synthesis. Continuous cellobiose-grown cultures maintained either at high dilution rates or with a high feed substrate concentration exhibited decreased cellulase synthesis; there was a large (sevenfold) decrease between 0 and 0.2 g of cellobiose per liter, and there was a much more gradual further decrease for cellobiose concentrations >0.2 g/liter. Several factors suggest that cellulase synthesis in C. thermocellum is regulated by catabolite repression. These factors include: (i) substantially higher cellulase yields observed during batch growth on Avicel than during batch growth on cellobiose, (ii) a strong negative correlation between the cellobiose concentration and the cellulase yield in continuous cultures with varied dilution rates at a constant feed substrate concentration and also with varied feed substrate concentrations at a constant dilution rate, and (iii) the presence of sequences corresponding to key elements of catabolite repression systems in the C. thermocellum genome.     

Induction of Cellulases in chaetomium cellulolyticum by cellobiose

The production of extracellular cellulases by Chaetomium cellulolyticum could be induced by slow feeding of cellobiose to the cultures. Both the rate of production and the amount of activity were comparable to that obtained in batch cultivation on cellulose. The specific filter paper activity of 2.06 U per mg protein was almost two times higher than that obtained in cellulose medium. Cellulases were not induced when glucose was slowly fed to the cultures. Changing the feed stream from glucose to cellobiose resulted in a rapid accumulation of cellulases. Thus cellobiose has a similar role in cellulase induction in C. cellulolyticum, as earlier shown for Trichoderma reesei.     

Enzymatic hydrolysis of cellulose to glucose lung immobilized β-glucosidase

The production of sugars by enzymatic hydrolysis of cellulose is a multistep process which includes conversion of the intermediate cellobiose to glucose by β-glucosidase. Aside from its role as an intermediate, cellobiose inhibits the endoglucanase components of typical cellulase enzyme systems. Because these enzyme systems often contain insufficient concentrations of β-glucosidase to prevent accumulation of inhibitory cellobiose, this research investigated the use of supplemental immobilized β-glucosidase to increase yield of glucose. Immobilized β-glucosidase from Aspergillus phoenicis was produced by sorption at controlled-pore alumina with about 90% activity retention. The product lost only about 10% of the original activity during an on-stream reaction period of 500 hr with cellobiose as substrate; maximum activity occurred near pH 3.5 and the apparent activation energy was about 11 kcal/mol. The immobilized β-glucosidase was used together with Trichoderma reesei cellulase to hydrolyze cellulosic materials, such as Solka Floc, corn stove and exploded wood. Increased yields of glucose and greater conversions of cellobiose of glucose were observed when the reaction systems contained supplemental immobilized β-glucosidase.     

A novel biochemical route for fuels and chemicals production from cellulosic biomass

The conventional biochemical platform featuring enzymatic hydrolysis involves five key steps: pretreatment, cellulase production, enzymatic hydrolysis, fermentation, and product recovery. Sugars are produced as reactive intermediates for subsequent fermentation to fuels and chemicals. Herein, an alternative biochemical route is proposed. Pretreatment, enzymatic hydrolysis and cellulase production is consolidated into one single step, referred to as consolidated aerobic processing, and sugar aldonates are produced as the reactive intermediates for biofuels production by fermentation. In this study, we demonstrate the viability of consolidation of the enzymatic hydrolysis and cellulase production steps in the new route using Neurospora crassa as the model microorganism and the conversion of cellulose to ethanol as the model system. We intended to prove the two hypotheses: 1) cellulose can be directed to produce cellobionate by reducing β-glucosidase production and by enhancing cellobiose dehydrogenase production; and 2) both of the two hydrolysis products of cellobionate--glucose and gluconate--can be used as carbon sources for ethanol and other chemical production. Our results showed that knocking out multiple copies of β-glucosidase genes led to cellobionate production from cellulose, without jeopardizing the cellulose hydrolysis rate. Simulating cellobiose dehydrogenase over-expression by addition of exogenous cellobiose dehydrogenase led to more cellobionate production. Both of the two hydrolysis products of cellobionate: glucose and gluconate can be used by Escherichia coli KO 11 for efficient ethanol production. They were utilized simultaneously in glucose and gluconate co-fermentation. Gluconate was used even faster than glucose. The results support the viability of the two hypotheses that lay the foundation for the proposed new route.     

Induction of Cellulase by Gentiobiose and Its Sulfur-Containing Analog in Penicillium purpurogenum

Cellulase induction by beta-glucodisaccharides was investigated by using non-cellulase-induced mycelia of Penicillium purpurogenum P-26, a highly-cellulase-producing fungus. Gentiobiose induced significant amounts of cellulase compared with cellobiose when nojirimycin was added to the induction medium to inhibit extracellular beta-glucosidase activity. Thiogentiobiose (6-S-beta-d-glucopyranosyl-6-thio-d-glucose), a sulfur-containing analog of gentiobiose, was more effective for cellulase induction than gentiobiose even in the absence of nojirimycin. Thiogentiobiose appeared to be a gratuitous inducer since it was not metabolized during cellulase induction. Gentiobiose was formed from cellobiose by the intracellular beta-glucosidase of P. purpurogenum. These findings indicate that gentiobiose is an active inducer of cellulase for this fungus and may possibly be formed by intracellular beta-glucosidase from cellobiose.     

Cellulase and beta-glucosidase production by a basidiomycete species.

The optimisation of cellulase and beta-glucosidase production by a basidiomycete species was studied and cellulase and cellobiase production by this and Trichoderma viride (and its mutants) in shake flasks were compared. The former produced an active cellulase comparable to that of T. viride when tested on filter paper, carboxymethylcellulose, and cotton; however, it produced 20 to 26 times larger amounts of cellobiase. Both cellulase and beta-glucosidase were obtained in good yield only when cellulose was the carbon source. The production of these enzymes was not repressed by readily assimilated carbon sources in the presence of cellulose. Only traces of cellulase and beta-glucosidase were formed on glucose, fructose, maltose, and cellobiose although good growth was obtained on these substrates. These enzymes were not induced on sophorose, lactose, mannitol, or glycerol and growth was poor on these substrates. Cellobiose octaacetate was a less effective inducer of cellulase and beta-glucosidase than was cellulose.